title: Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein alpha-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not.
abstract: Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.
authors.affiliation: Department of Anaerobic Microbiology, Virginia Polytechnic Institute, State University, Blacksburg 24061.
authors.forename: D R
authors.initials: DR
authors.lastname: Dean
authors.forename: R A
authors.initials: RA
authors.lastname: Setterquist
authors.forename: K E
authors.initials: KE
authors.lastname: Brigle
authors.forename: D J
authors.initials: DJ
authors.lastname: Scott
authors.forename: N F
authors.initials: NF
authors.lastname: Laird
authors.forename: W E
authors.initials: WE
authors.lastname: Newton
mesh_terms: D000595:Amino Acid Sequence; D001395:Azotobacter / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D008983:Molybdoferredoxin; D006639:Histidine; D009591:Nitrogenase; D003545:Cysteine